Research And Application Of Affinity Extraction Technology For The Detection Of Antibiotic Residues In Aquatic Products

Antibiotic residues in aquatic products has been a threat to food safety and public health. Thereare a lot of kinds methods for detection of antibiotic residues, The most common way ismicrobiological assay method, high performance liquid chromatography （HPLC） high performanceliquid chromatography-mass spectrometry （HPLC-MS） and so on. These methods are appropriatefor qualitative and quantitative detection in terms of antibiotic residues. Due to the complexity ofmatrix of aquatic products sample, the key to the analysis of drug residues in aquatic products issample preparation. Pretreatment methods such as liquid-liquid extraction, solid phase extraction（SPE） and matrix solid-phase dispersion method （MSPD）, are cumbersome in operation, which areeasy to cause loss of drug residues. So the exist form of antibiotic residues in main matrix is theprecondition of development of pretreatment methods.Protein is the main substrate of fish samples in antibiotic residues detection, Many studies haveshown that antibiotics has strong binding with protein, which can reduce the extraction efficiency ofantibiotics. The effect of protein on drug extraction efficiency have yet to attract the attention ofresearchers, therefore, protein binding with antibiotics should attract the attention of researchers, andhow the drug was released from the complex substrate become important.In addition, chemical structure’s changes of antibiotics in different pH condition will also affectthe extraction efficiency of drugs.This paper aim to research the combination of antibiotics and protein function by fluorescencequenching method, clarify the exist state of drugs in the sample matrix of aquatic products, to providetheoretical basis for the extraction of drug residues. Combined with the improved QuEChERSmethod, acetonitrile-inorganic salt-water, weak acid extraction and liquid chromatographic， weestablished detection technology on tetracycline drugs, sulfa drugs, methods of quinolones in fishsamples.The paper can be divided into five sections:Part one: In this paper, the pretreatment methods of complex samples were reviewed, and thebackground and content and significance of this issue were introduced.Part two：The fluorescence quenching method and HPLC were applied to study the form of sulfonamides （SAs） and trimethoprim （TMP） in protein matrix of aquatic products. Meanwhile, theinfluence of the protein on extraction of SAs and TMP was investigated in detail. The results showedthat SAs and TMP strongly bound with FSA. In80%（V/V） tetrahydrofuran aqueous solution, a slowbut full protein denaturation takes place, which would cause the unfolding of protein and release ofdrug residues. When K2HPO4was added to the extractant of tetrahydrofuran solution，the aqueoustwo phase system （ATPS） was formed. The liposoluble drug residues were extracted to the upperphase of ATPS with high extraction efficiencies. Meanwhile, the fat-soluble impurities are extractedless in80%（V/V） tetrahydrofuran aqueous solution than those of in hydrophobic solvents asliquid-liquid extraction procedure. Therefore the purification steps were greatly simplified. Overallrecoveries were80.9～95.1%with RSD values of1.9⁵.9%, and the detection limits were in therange of6.3～23.7ng/g. The proposed approach was satisfactorily applied to the quickdetermination of SAs and TMP residues in fish.Part three：In this paper, molecular fluorescence spectrometry and High performance liquidchromatography（HPLC） were applied to study the form of tetracyclines （TCs） in protein matrix ofaquatic products. The molecular form of TCs in different media of pH value was studied by UV-VIS.The influence of proteins on extraction of TCs from aquatic products samples was also discussed.The results showed that there was a strong binding effect between TCs and protein. In the mixture ofammonium sulfate aqueous solution and mobile phase of HPLC（methanol: acetonitrile:0.02mol/Loxalic acid aqueous solution=20:20:60）, a slow but full protein denaturation takes place, whichcauses the dissociation of TCs-protein complex. Therefore, TCs residues in aquatic products wereextracted with high extraction efficiencies. This method has been applied to the determination of4kinds of TCs （oxytetracycline, tetracycline, chlortetracycline and doxycycline） residues in the fishand shrimp samples. The detection limits were in the range of14.8～39.2ng/g, and the spiked recoveries were88.2%～95.4%with RSD1.3%～4.9%. The method was simple, quick andreliable.Part four：In this paper, rayleigh scattering fluorescence spectrometry and High performanceliquid chromatography（HPLC）were applied to study the form of Quinolones （QNs） in proteinmatrix of aquatic products and the changes of protein structure in different concentrations weak acid.The influence of proteins on extraction of QNs from aquatic products samples was also discussed.There was a strong binding effect between QNs and protein. In the0.5mol/L citric acid watersolution, a slow but full protein denaturation takes place, which causes the dissociation of QNs-protein complex. Therefore, QNs residues in aquatic products were extracted with high extractionefficiencies. This method has been applied to the determination of6kinds of QNs （enoxacin,norfloxacin, Ciprofloxacin, lomefloxacin, enrofloxacin and gatifloxacin） residues in the fish. Thedetection limits were in the range of14.8³9.2ng/g, and the spiked recoveries were81.5%⁹6.2%with RSD1.9%⁵.9%. The method was simple, quick and reliable.Part five：In this study, a simple and effcient acetonitrile-sodium chloride aqueous two-phasesystem（ATPS） is developed coupled with inductively coupled plasma atomic emissionspectrometry（ICP-AES）, for the extraction and the determination of cooper and lead in salt. Samplesare pretreated by ATPS with1-Pyrrolidinecarbodithioic acid ammonium salt （APDC） used ascomplex agent. The phase separation conditions and extraction effciency factors, such as pH andmass fraction of acetonitrile are investigated and optimized. The recoveries of copper and lead are106%and96.2%with the RSD are0.76%and0.83%respectively. And limits of detection are5.0×10^-4mg/kg and2.3×10^-4mg/kg respectively. The proposed method is successfully applied forthe preconcentration and determination of copper and lead in real salt samples. It shows that themethod can remove the interference of salt matrix when determine Cu and Pb by ICP-AES.